Inhibition of SUMO‐mediated Protein‐Protein Interactions in DNA Damage Repair

Abstract

Maintenance of genomic integrity requires functional repair mechanisms. Some mechanisms include the use of sumoylation, a process in which proteins are post‐translationally modified with a small peptide SUMO (small ubiquitin‐like modifier). In response to DNA damage, multiple proteins become sumoylated. Current evidence suggests that sumoylation in this context mediates protein‐protein interactions to promote DNA damage repair. SUMO‐interacting motifs (SIMs) play an essential role in SUMO‐mediated protein‐protein interactions. The SIM on a protein is a region that allows it to recognize and bind the SUMO attached to another protein. Previous studies have shown that inhibiting this type of interaction can lead to sensitized cancer cells by impairing DNA damage repair and response. Thus, this project aims to study a similar inhibition by overexpressing SIMs naturally found in two proteins, Slx5 and Sgs1, in the eukaryotic model organism, Saccharomyces cerevisiae. Both proteins are a part of their respective protein complexes, and their SIMs serve as a functional way for the complexes to interact with sumoylated proteins during DNA repair. We hypothesize that overexpressed SIMs will disrupt endogenous SUMO‐SIM interactions important for DNA damage repair and that cells with overexpression of the SIM would be sensitized to DNA damaging agents. We observed that cells overexpressing SIMs from Slx5 exhibited a growth defect compared to cells without overexpression. However, when treated with DNA damaging agents, such as methyl methanesulfonate and UV, cells with SIM overexpression exhibited little to no sensitivity compared to the wild type.To further explore the effect of SUMO‐SIM interaction, we have designed another protein containing the DNA‐binding (SAP) domain of Siz2, a SUMO protein ligase specific to DNA damage, and the SUMO‐binding domain of Ulp1, a SUMO isopeptidase, to potentially disrupt SUMO:SIM interactions involved in DNA repair. Inclusion of the SAP domain along with a nuclear localization signal (NLS) is hypothesized to increase the nuclear localization of the protein during DNA repair, which will be observed using fluorescent microscopy of a GFP tag incorporated into the protein. With increased nuclear localization and inclusion of the SUMO‐binding domain, we hypothesize that there will be disruption to the SUMO:SIM interactions involved in DNA damage repair, resulting in an increase in the cells’ sensitivity to DNA damaging agents.